Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

Abstract

Background: Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity.

Results: High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site.

Conclusions: Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

Figure 1

HRM analysis of ETFDH gene. Electropherograms of various mutations: (A) c.250G > A (wild type); (B) c.250G > A (heterozygous); (C) c.383T > C (wild type); (D) c.383T > C (heterozygous); (E) Normalized and temperature-shifted difference plots, the melting profile of c.250G > A; (F) Normalized and temperature-shifted difference plots, the melting profile of c.383T > C; (G) The mutation of c.383T > C was not detected in 60 unrelated control chromosomes (wild type); (H) Affected pedigree with familial segregation of both mutations. Squares, male subjects; circles, female subjects. Affected and unaffected subjects are represented by solid and open symbols, respectively. The subject with a sample available for the present study is indicated with an asterisk.

Figure 2

Structure prediction of ETF:QO. (A) The predicted wild-type model of human ETF:QO. The structure comprises three domains: the FAD-binding domain (gray), the ubiquinone-binding domain (blue) and the 4Fe4S cluster domain (red). The three redox centers: FAD, ubiquinone and the 4Fe4S cluster are shown in ball-and-stick representations. A cluster of hydrophobic residues which are located at the FAD-binding domain and at a distance below 4 Å around the mutation residues (p.Ala84Thr and p.Phe128Ser) are depicted in the sphere model. (B) A close-up view of the hydrophobic residues (V71, I73, A84, V85, L87, V100, L127 and F128). The α-helices and β-strands are also labeled. (C) Model of the p.Ala84Thr mutant in which the hydrophobic residue A84 is replaced by the hydrophilic residue threonine (yellow). (D) Model of the Phe128Ser mutant in which the hydrophobic residue F128 is replaced by the hydrophilic residue serine (yellow). The 3D molecular graphs are displayed using PyMOL [51].

Figure 3

RMSD plots of the FAD-binding motif. Comparison of the RMSD plots of the FAD-binding motif (residues 71-90) of WT and MT (A) p.Ala84Thr and (B) p.Phe128Ser structures with respect to the starting conformation during the course of the simulation. WT and MT plots are presented in blue and red, respectively.

Figure 4

B-factors analysis. Structures of the (A) WT, (B) p.Ala84Thr and (C) p.Phe128Ser ETF:QO proteins are drawn in cartoon putty representation at the β1, α1, α2, and α3 regions, where the color is ramped by residue from blue as the lowest B-factor value to red as the highest B-factor value. In addition, the size of the tube also reflects the value of the B-factor, where the larger the B-factor the thicker the tube. The structures in the other regions are colored in white and displayed in cartoon tube representation, where the size of the tube is independent of the B-factors. The red arrows indicate the positions of the mutations.

Figure 5

Cross-correlation map calculated from the NMA. A negative value refers to an anti-correlation between residue fluctuations (blue), whereas a positive value indicates concerted motion in the same direction (red).